ABSTRACT Mitochondria are essential subcellular organelles that serve multiple functions in cellular biology including energy production, metabolism, lipid synthesis and signaling. Due to the ease of genetic manipulation in mouse models, a large majority of mechanistic inferences about heart failure have been made from gain of function or loss of function studies of specific enzymes during heart failure in mice. However, profound differences in the chemistry and physiology of murine mitochondria are present in comparison to human mitochondria. These include vast differences between murine and human mitochondrial phospholipid compositions, their repertoires of lipid 2nd messengers generated in response to stress, and the enzymic mediators responsible for modulating human mitochondrial signaling and bioenergetics. To bridge the translational gap between mouse and humans, we have focused our studies on the roles of lipid 2nd messengers present in human hearts and their impact on human heart mitochondrial bioenergetic and signaling functions. Recently, we have discovered a series of novel lipid 2nd messengers generated by human mitochondria which are dynamically regulated during the progression of heart failure in humans. Moreover, we have demonstrated that these novel lipid 2nd messengers modulate human mitochondrial energy production, bioenergetic efficiency and cellular signaling. Accordingly, a major goal of the proposed research is to identify the chemical mechanisms that integrate mitochondrial function with human cardiac myocyte physiology through novel lipid 2nd messengers we identified in human myocardium. Through utilization of an integrated series of mass spectrometric technologies we developed/employ including LC-MS/MS with charge-switch derivatization, multiple reaction monitoring, and high mass accuracy analysis of suites of product ions, we have identified and quantified many new natural products some of which we have already been shown to modulate human mitochondrial bioenergetics and signaling. The first goal is to identify the molecular mechanisms which initiate the generation of these novel lipid 2nd messengers from human myocardial phospholipids and identify the biochemical pathways which generate these natural products. The second goal of the proposed research is to identify the chemical diversity of these natural products and clarify their enantiomeric purity in extracts of human myocardium. Identification of enantiomerically pure natural products in human myocardium will demonstrate that they are produced by specific enzymes and that they are not products of adventitious oxidation. The third goal of the proposed studies is to mechanistically identify their roles in modulating human heart mitochondrial bioenergetics and signaling. Collectively, the proposed research is a unique opportunity to dramatically accelerate identification of the roles of human heart lipid 2nd messengers in precipitating human mitochondrial dysfunction that promotes the progression of human heart failure. This targeted translational approach will directly identify bona fide pharmacologic targets for treatment of human heart failure.